Summary Inhibition of secretion by Gi/o-coupled GPCRs is an important control mechanism used by many hormones and neuromodulators. It is well documented that activation of Gi/o-coupled GPCRs in secretory cells releases G?? subunits that inhibit Ca2+ entry through voltage-dependent Ca2+ channels (VDCCs), leading to reduced hormone release. However, a direct interaction between G?? and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins also inhibits transmitter hormone release in many systems. This mechanism is not only more acute and direct in controlling evoked release but also has the ability to modify spontaneous release. However, the mechanistic details of this SNARE-mediated modulation remain understudied, with many open questions. For example, which GPCRs work through this mechanism? We have also found that G?? inhibition of Ca2+ entry synergizes with G?? inhibition of SNARE-mediated exocytosis. We will address the mechanism of this synergism as well as it?s implications in physiology. Adding another layer of complexity is the diversity of G? and G? isoforms and the control of specificity of G??s. Using proteomic assays, we have found that specific G?? subunits (i.e. G?1?2) bind to SNARE even without GPCR agonists whereas adding agonists enhances G?1?2 binding but also brings new G??s (e.g. G?2?3) to SNAREs. These data suggest that unique G?? subunits can differentially act on SNARE to achieve different degrees of modulation via GPCRs or even without GPCR activation. Therefore, we propose that SNARE-mediated G?? modulation of hormone release exerts its functional diversity by different combination of G?? subunits and different degree of SNARE binding. This leads us to focus on three specific aims that test (1) which GPCRs work through modulation of Ca2+ entry and which work through binding SNARE (2) what is the mechanism of synergism between the two G??-mediated mechanisms and (3) what is the role of particular G? and ? subunits in GPCR regulation of hormone release. Given the huge diversity of GPCRs in CNS, given G???s close ties to modulation of exocytosis, and given their relevance to many hormonal and neurological disorders, this project will illuminate a more versatile modulation of secretion by different G??s, bridge the knowledge gap between tonic and phasic modulation of release via GPCRs in secretion, and unravel molecular mechanisms underpinning various hormonal and neurological disorders.